Characterization of organic fluorophores forin vivoFRET studies based on electroporated molecules

Abstract

In vivo single-molecule fluorescence and Förster resonance energy transfer (FRET) techniques are excellent tools for studying spatial distribution, the nanoscale structure and conformational changes in living cells. We have recently introduced an electroporation-based method to internalize DNA and proteins labeled with organic fluorophores into living bacteria and established the ability for long-lived single-molecule fluorescence and FRET measurements. However, further developments, such as optimization of electroporation conditions, evaluation of organic fluorophore performance in vivo and quantitative single-cell FRET analysis, are needed to make the method more robust and general. Using singly-labeled DNA fragments, we optimized internalization efficiency and cell viability at six electroporation voltages, achieving >60% loading and viability similar to non-treated cells. We characterized the photostability and brightness of three donor fluorophores and four acceptor fluorophores in vivo; Cy3B, Atto647 and Atto647N performed best with photobleaching lifetimes of ∼20 s, 46 s and 92 s, respectively, and brightness values of ∼4000 photons per second under the same illumination conditions. We used three doubly-labeled DNA FRET standards (having in vitro FRET efficiencies of ∼17%, ∼42%, and ∼88%) and an alternating-laser excitation scheme to measure apparent FRET efficiencies at the single-cell level. We showed that we could differentiate DNA FRET standards at the single-cell level. Our approach offers a powerful method for the study of intramolecular changes or complex formation using FRET at the single-cell level in live bacteria.

Illuminating Messengers: An Update and Outlook on RNA Visualization in Bacteria

“…These techniques are not (yet) compatible with the small-sized and cell wall-enclosed bacteria. Electroporation can be used to internalize short DNA fragments or proteins up to 100 kDa in E. coli while at the same time keeping cells in a viable state (Plochowietz et al, 2014; Sustarsic et al, 2014; Aigrain et al, 2015; Di Paolo et al, 2016). It would be of great interest to elucidate whether fluorescently labeled FIVH probes, such as molecular beacons or LIT probes, could be introduced in bacteria in a similar fashion.…”

Unveiling the Inner Workings of Live Bacteria Using Super-Resolution Microscopy

“…144,145 The major advantage of this approach is that it allows proteins to be labeled with small molecule dyes and purified outside the cell. While this approach is very promising, a number of possible disadvantages must be addressed before it can be widely used.…”

Single Pair Förster Resonance Energy Transfer: A Versatile Tool To Investigate Protein Conformational Dynamics

“…Schematics of exemplary protein conformational dynamics are shown in Figure A. Recently, spFRET has also been expanded to studies in cellular systems …”

“…Recently, spFRET has also been expanded to studies in cellular systems. [12,13] Proteins for in vitro experiments are expressed and purified in the same way as for other biochemical or biophysical experiments with the great advantage that only a small amount of protein is required for performing the experiments. Following the purification, the protein is fluorescently labeled by the attachment of donor and acceptor fluorophores.…”